Interaction of Neutrophils with Macrophages Promotes IL-1β Maturation and Contributes to Hepatic Ischemia–Reperfusion Injury

Hepatic ischemia–reperfusion (I/R) injury is a major cause of liver dysfunction and serious complications in hepatic surgery and liver transplantation. Systemic low-flow ischemia and hypoxia, such as trauma, hemorrhagic shock, sepsis, congestive heart failure, and respiratory failure, may also lead to hepatic I/R injury. One prominent feature of hepatic I/R injury is excessive inflammatory response characterized by the release of inflammatory cytokines and chemokines that recruit circulating leukocytes, mainly neutrophils, into the ischemic tissues (1). The recruited neutrophils may subsequently lead to dramatic hepatic I/R injury. Indeed, experimental studies have shown that the inhibition of the inflammatory response by neutrophil depletion or through neutralizing Abs against chemokines for neutrophils can reduce hepatic I/R injury (2). However, the mechanism by which I/R stimuli trigger an inflammatory response after I/R in the liver is not been fully understood.

Recent evidence indicates that inflammation in the absence of pathogens, which is referred to as sterile inflammation, is mediated through the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasomes—intracellular, large multiple-protein complexes that regulate the maturation of a potent proinflammatory cytokine IL-1β (3). NLRP3 inflammasomes contain NLRP3, adaptor molecule apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and cysteine protease caspase-1, which induce caspase-1 activation. Because caspase-1 is an IL-1β–converting enzyme, its activation processes pro–IL-1β into its mature form and induces IL-1β release, thereby leading to inflammatory response and tissue injury. Indeed, we have recently demonstrated the importance of NLRP3 inflammasomes in the pathogenesis of sterile inflammation-related diseases (4–8). We have also revealed that NLRP3 possesses inflammasome-independent functions during hepatic I/R injury (9). In particular, NLRP3 deficiency impairs neutrophil accumulation in the I/R liver and attenuates hepatic I/R injury independently of the inflammasomes, a process accompanied by a reduction of IL-1β release. These findings allow us to hypothesize that this inflammasome-independent IL-1β release contributes to the development of hepatic I/R injury.

To test this hypothesis, we used IL-1β–deficient (IL-1β^−/−) and neutrophil- or macrophage-depleted mice, and found that these mice showed limited inflammatory response and tissue injury after hepatic I/R. In vitro experiments revealed that neutrophil-derived proteases induced maturation of pro–IL-1β derived from macrophages. Furthermore, inhibition of neutrophil serine proteases attenuated IL-1β release and hepatic I/R injury. These findings demonstrate that the interaction between neutrophils and macrophages plays a crucial role in IL-1β–driven inflammatory response during hepatic I/R injury, and provide new insights into the inflammasome-independent mechanism of IL-1β maturation in the liver.

All experiments in this study were performed in accordance with the Jichi Medical University Guide for Laboratory Animals. Wild type (WT) and IL-1β^−/− mice (male, 8–12 wk old, C57BL/6J background) were purchased from SLC Japan (Shizuoka, Japan) and kindly provided by Dr. Y. Iwakura (Tokyo University of Science, Chiba, Japan) (10). Mice were housed in an environment maintained at 23 ± 2°C with free access to food and water under a 12 h light and dark cycle with the lights on from 7:00 to 19:00. The mice underwent hepatic I/R surgery or sham operations. Partial hepatic ischemia was produced as previously described (9). Mice were anesthetized with isoflurane. Midline laparotomy was performed, and an atraumatic clip (Fine Science Tools, Foster City, CA) was placed across the portal vein, hepatic artery, and bile duct to interrupt blood supply to the left lateral and median lobes (∼70%) of the liver. After 60 min of partial hepatic ischemia, the clip was removed to initiate reperfusion. Sham control mice underwent the same protocol without vascular occlusion. Mice were sacrificed at the indicated periods of reperfusion, and samples of blood and ischemic lobes were collected for analysis. To examine the effect of serine proteinase inhibition on hepatic I/R injury, mice were treated s.c. with 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF; 10 mg/kg; Roche Diagnostics, Indianapolis, IN) or saline (control) 10 min before ischemia, and 10 min before, then 1 and 3 h after reperfusion. All efforts were made to minimize animal discomfort and suffering.

Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase, total bilirubin, blood urea nitrogen, and creatinine were measured using the chemical analyzer Fuji-DRYCHEM (Fuji Film, Tokyo, Japan) according to the manufacturer’s instructions.

IL-1β, IL-6, TNF-α, and CCL2 levels were assessed using a mouse ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

Histology and immunohistochemistry were performed as described previously (9). The paraffin-embedded tissue sections were stained with H&E. The injured area was quantified in 10 randomly high-power fields per section (original magnification ×400). The severity of liver injury was graded according to Suzuki Score Criteria on a scale from 0 to 4 (11). No necrosis or congestion and centrilobular ballooning was given a score of 0. Severe congestion and ballooning degeneration, as well as >60% lobular necrosis were given a value of 4. Immunohistochemical analysis was performed using the following Abs: oxidative stress marker 4-hydroxy-2-nonenal (4-HNE; clone HNEJ-2; Japan Institute for the Control of Aging, Nikken SEIL, Shizuoka, Japan), macrophage marker CD68 (AbD Serotec, Kidlington, U.K.), and neutrophil marker granulocyte receptor-1 (Gr-1) (eBioscience, San Diego, CA). Isotype-matched IgG (Vector Laboratories, Burlingame, CA) was used as a negative control. The stained sections were digitalized and analyzed using a microscope (FSX-100; Olympus, Tokyo, Japan).

Apoptotic cells were identified using an In situ Apoptosis Detection Kit (Takara Bio, Shiga, Japan) using the TUNEL method.

Total RNA was prepared using ISOGEN (Nippon Gene, Toyama, Japan) according to the manufacturer’s instructions. Real-time RT-PCR analysis was performed using the Thermal Cycler Dice Real-Time System II (Takara Bio) to detect the mRNA expression of Il1b, Il6, Tnfa, Ccl2, Cxcl1, Cxcl2, Ifng, Il18, Il1rn, and Hprt. The following primers were used: Il18 (5′-CAGGCCTGACATCTTCTGCAA-3′ and 5′-TCTGACATGGCAGCCATTGT-3′), Il1rn (5′-GCTCATTGCTGGGTACTTACAA-3′ and 5′-CCAGACTTGGCACAAGACAG-3′), and Hprt (5′-TGAAGGAGATGGGAGGCCA-3′ and 5′-AATCCAGCAGGTCAGCAAAG-3′). Other primers have been described previously (9). The expression levels of each target gene were normalized by subtracting the corresponding Hprt threshold cycle value; normalization was carried out using the ΔΔ threshold cycle comparative method.

Livers were perfused with ice-cold PBS, minced, incubated with 2 mg/ml collagenase P (Roche, Basel, Switzerland) solution for 20 min, and filtered through a 70-μm nylon mesh strainer. Nonparenchymal cells were separated from hepatocytes by centrifuging at 2000 × g for 5 min three times. Fc-γ receptors were blocked with rat anti-mouse CD16/CD32 Ab (Fc-γ III/II receptor; BD Biosciences) for 15 min on ice. Cells were then stained with the following Abs for 20 min in the dark: allophycocyanin-conjugated anti-CD45 (eBioscience), FITC-conjugated anti-CD45R (BD Biosciences), PE-conjugated anti-F4/80 (eBioscience), FITC-conjugated anti-CD11b (BD Biosciences, Franklin Lakes, NJ), and PE-conjugated anti-Ly6G (BD Biosciences). The cells were washed with 0.1% BSA in PBS and subjected to flow cytometric analysis (FACSVerse; Becton Dickinson, NJ). Dead cells were excluded on the basis of 7-AAD staining, then leukocytes were selected by their expression of CD45. Macrophages and neutrophils were identified by the expression of CD45⁺CD11b⁺F4/80⁺ and CD45⁺Ly6G⁺CD45R⁻, respectively. The analysis was performed using FlowJo software version 10 (Tree Star, San Carlos, CA). Isotype-matched IgG was used as a negative control to exclude nonspecific background staining.

For macrophage depletion, mice were treated intravenously with clodronate liposome or control liposome (150 μl/mice; FormuMax Scientific, Sunnyvale, CA) 24 h before hepatic I/R. For neutrophil depletion, mice were treated i.p. with anti–Gr-1 mAb (clone RB6-8C5; kindly provided by Dr. R. Coffman, DNAX Research Institute) (12) or isotype control IgG (Jackson ImmunoResearch, West Grove, PA) 24 h before hepatic I/R. To assess depletion of macrophages or neutrophils, blood or liver samples were obtained 24 h after the injection, and the percentages of F4/80⁺CD11b⁺ (macrophages) or Ly6G⁺CD45R⁻ cells (neutrophils) were analyzed by flow cytometry.

Murine peritoneal macrophages were isolated using the thioglycollate elicitation method and cultured in RPMI 1640 (WAKO, Osaka, Japan) supplemented with 10% FCS. Murine bone marrow–derived neutrophils were isolated by negative selection using the Neutrophil Isolation Kit (Miltenyi Biotec, Tokyo, Japan) and cultured in 10% FCS/RPMI 1640. Murine primary hepatocytes and nonparenchymal cells were isolated using a collagenase perfusion method as previously described (9). To further purify Kupffer cells, nonparenchymal cells were cultured for 2 h and attached onto the plastic surface. After rinsing with PBS to remove nonadherent cells, attached Kupffer cells were harvested with high purity (≥90%) as assessed by F4/80 staining (13). Human neutrophils from peripheral blood were isolated using a standard technique (14). After dextran sedimentation and hypotonic lysis of the remaining erythrocytes, granulocytes were separated by Ficoll-Paque PLUS (GE Healthcare Biosciences) density gradient centrifugation. All reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise specified.

To ensure the induction of pro–IL-1β, macrophages were primed with a low dose of LPS (100 ng/ml) for 10 h and subsequently cocultured with various numbers of neutrophils (macrophage/neutrophil ratio, 1:0–1:8) for 4 h, and IL-1β levels in the supernatants were assessed. To determine the cell type responsible for IL-1β release, neutrophils and macrophages isolated from IL-1β^−/− mice were used. For blocking experiments, both neutrophils and macrophages were pretreated with the indicated concentrations of caspase-1 inhibitor (Z-YVAD-FMK; BioVision, San Francisco, CA), neutrophil elastase inhibitor (NEI, Elastase Inhibitor II; Calbiochem, San Francisco, CA), or serine proteinase inhibitor AEBSF for 1 h.

Western blotting was performed as previously described to detect IL-1β (7). Primary Abs against IL-1β (R&D Systems) and anti-M2 FLAG (Sigma) were used. Immunoreactive bands were visualized by Western BLoT Quant HRP Substrate (Takara Bio).

The mutated IL-1β_V114E, IL-1β_Y113D, and IL-1β_D116I were generated using the PrimeSTAR Mutagenesis Basal kit (Takara Bio) and subcloned into a CS-CA-MCS plasmid (kindly provided by Dr. H. Miyoshi, RIKEN BioResource Center, Ibaraki, Japan) (15) with C-terminal FLAG-6× His tag. To prepare the lentiviral vectors, HEK293T cells were transfected with CS-CA-MCS, pLP1, pLP2, and pVSVG using PEI MAX (Polysciences, Warrington, PA). Culture media containing the lentiviral vectors were collected and purified by ultracentrifugation. The lentiviral titer was measured using a Lentivirus qPCR Titer kit (Applied Biological Materials, Richmond, BC, Canada). For production of THP-1 cells expressing mutated IL-1β, cells were transduced in the presence of 8 μg/ml polybrene (Sigma-Aldrich). Transfected FLAG–IL-1β proteins were purified with Flag-M2 beads from cell lysates. The concentrated FLAG–pro–IL-1β proteins (2 mg) were incubated with human neutrophils (2 × 10⁶ cells) for 3 h and analyzed by Western blotting.

Data are expressed as the mean ± SEM. An unpaired t test was used for comparisons between two groups. For comparisons between multiple groups, the significance of the difference between means from the groups was determined by one-way ANOVA combined with a post hoc test. A p value <0.05 was considered to be statistically significant.

To investigate the role of IL-1β in hepatic I/R injury, we initially assessed serum and hepatic levels of IL-1β in a murine model of hepatic I/R. Consistent with previous reports (9, 16), the levels of serum IL-1β protein and hepatic Il1b expression were clearly elevated 6 h after hepatic I/R (60 min ischemia followed by reperfusion) (Fig. 1A, 1B), indicating the role of IL-1β in the development of hepatic I/R injury. To explore its contribution, we subjected IL-1β^−/− mice to hepatic I/R and found that the injured liver area determined by HE staining was significantly decreased in IL-1β^−/− mice compared with WT mice (Fig. 1C, 1D). Similarly, pathological severity was assessed based on Suzuki Score Criteria and also showed improvement in IL-1β^−/− mice (Fig. 1E). In addition, IL-1β^−/− mice showed lower serum ALT levels after 6 h of reperfusion compared with WT mice (Fig. 1F).

We subsequently performed real-time RT-PCR analysis to assess the expression of inflammatory cytokines. The expression of Il6, Tnfa, Ccl2, Cxcl1, and Cxcl2 was markedly increased in the I/R liver of WT mice, whereas the increased expression of these cytokines was significantly decreased in IL-1β^−/− mice (Fig. 2). A similar expression pattern of Ifng was observed. Expression of Il1rn, but not Il18, was increased after hepatic I/R, but there was no significant difference of Il18 and Il1rn expression between WT and IL-1β^−/− mice. Consistent with this finding, serum levels of IL-6, TNF-α, and CCL2 were significantly increased after hepatic I/R in WT mice, whereas the increased levels of these cytokines were decreased in IL-1β^−/− mice (Supplemental Fig. 1). These findings suggest that IL-1β deficiency failed to induce the expression of inflammatory cytokines, which may contribute to the development of hepatic I/R injury.

I/R initiates reactive oxygen species (ROS) generation, leading to apoptotic and necrotic cell death in the liver (1). We performed immunohistochemical analysis using 4-HNE and TUNEL staining to assess ROS generation and apoptosis, respectively. A number of 4-HNE⁺ cells were clearly visualized in the I/R liver of WT mice (Fig. 3A). However, the number of these cells was markedly lower in the I/R liver of IL-1β^−/− mice than in WT mice. TUNEL⁺ cells were also detected in the injured area of the I/R liver, and they were significantly decreased in the liver of IL-1β^−/− mice (Fig. 3A, 3B). Furthermore, we determined the infiltration of inflammatory cells by immunohistochemical analysis for CD68 (macrophages) and Gr-1 (neutrophils). As expected, the number of macrophages (CD68⁺ cells) and neutrophils (Gr-1⁺ cells) was increased in the liver of WT mice 6 h after I/R, whereas the increased infiltration of both cells was inhibited in the liver of IL-1β^−/− mice (Fig. 3C, 3D). These findings suggest that macrophages and neutrophils contribute to IL-1β–driven inflammation, ROS generation, and apoptosis during hepatic I/R injury.

Macrophages are the main cellular source of IL-1β. Thus, we investigated the role of macrophages in tissue injury and IL-1β release after hepatic I/R.

To deplete macrophages in vivo, we intravenously injected clodronate liposome or control liposome into mice. We confirmed that the number of macrophages (CD45⁺CD11b⁺F4/80⁺ cells) was almost completely depleted in the liver of clodronate liposome-injected mice (Fig. 4A). Depletion of macrophages significantly attenuated liver injury (injured area and Suzuki Score Criteria ), serum ALT levels, and the number of TUNEL⁺ cells after hepatic I/R (Fig. 4B–F). Furthermore, serum IL-1β levels were also significantly decreased after hepatic I/R in macrophage-depleted mice (Fig. 4G). Supporting this finding, real-time RT-PCR analysis showed that the increased expression of Il1b and Il6 in the I/R liver was significantly reduced in macrophage-depleted mice (Fig. 5). Similar changes were observed in the expression of Tnfa, Cxcl2, Ccl2, and Ifng. However, macrophage depletion did not influence the expression of Il18 and Il1rn after hepatic I/R.

We also examined the effects of neutrophil depletion using an anti–Gr-1 neutralizing Ab (RB6-8C5) in hepatic I/R injury. Intraperitoneal injection of anti–Gr-1 Ab completely depleted neutrophils (CD45⁺Ly6G⁺CD45R⁻ cells) in the liver (Fig. 6A) and peripheral circulation (data not shown). Similar to the data on macrophage depletion, the aforementioned manifestations, including liver injury, apoptosis, and IL-1β release, tended to be attenuated in neutrophil-depleted mice (Fig. 6B–G). Furthermore, the upregulation of Il1b, Il6, Tnfa, Cxcl2, and Il1rn in the I/R liver was also reduced in neutrophil-depleted mice (Fig. 7). These results indicate that both macrophages and neutrophils play an important role in the release of IL-1β and the development of hepatic I/R injury.

Primary murine peritoneal macrophages and bone marrow–derived neutrophils were prepared and cocultured to investigate the role of macrophages and neutrophils in IL-1β release. To avoid the activation, neutrophils were isolated using negative immune-magnetic selection (purity >90%) (Fig. 8A). Because IL-1β release requires pro–IL-1β synthesis, we primed the macrophages with a low dose of LPS, as previously described (4), and assessed IL-1β levels in the coculture supernatants. IL-1β levels were significantly increased by the coculture in a neutrophil number–dependent manner (Fig. 8B). Interestingly, a significant release of IL-1β was observed in a ∼1:4 macrophage/neutrophil ratio. We confirmed the processing of IL-1β by Western blotting (Fig. 8C). To determine the cell type responsible for IL-1β production, we prepared neutrophils or macrophages isolated from IL-1β^−/− mice and cocultured with macrophages and neutrophils from WT mice, respectively. Coculture of IL-1β^−/− neutrophils and WT macrophages stimulated IL-1β release, whereas coculture of WT neutrophils with IL-1β^−/− macrophages failed to do so (Fig. 8D), indicating that macrophages are responsible for the release of IL-1β. Moreover, these findings also suggest that neutrophil-derived proteases may induce the processing of pro–IL-1β derived from macrophages. To confirm this, we tested the effect of neutrophil protease inhibitors, such as AEBSF and NEI, on IL-1β release by coculture of neutrophils and macrophages. Coculture-induced IL-1β release was significantly inhibited by treatment with AEBSF (Fig. 8E). NEI treatment also decreased IL-1β release; however, the difference did not reach statistical significance (Fig. 8F). In contrast, treatment with the caspase-1 inhibitor Z-YVAD had no effect on IL-1β release (Fig. 8G). These findings indicate that neutrophil-induced macrophage IL-1β release is dependent on neutrophil-derived proteases and independent of inflammasomes.

To explore the cleavage site(s) of IL-1β by neutrophil proteases, three human IL-1β mutants were constructed: IL-1β_D116I, IL-1β_V114E, and IL-1β_Y113D (Supplemental Fig. 2A). Caspase-1 has been shown to cleave pro–IL-1β between Asp116 and Ala117. Indeed, we confirmed that the well-known NLRP3 inflammasome activator ATP failed to process IL-1β_D116I (data not shown). Although incubation with human neutrophils induced the maturation of WT and all mutant IL-1β, the bands of mature IL-1β were less intense in the cells transfected with IL-1β_V114E and IL-1β_Y113D than those in the cells transfected with IL-1β_WT and IL-1β_D116I (Supplemental Fig. 2B), suggesting that neutrophil proteases may cleave at least two sites, between Val114 and His115 and betweenTyr113 and Val114. Because the live-resident macrophages, Kupffer cells, are responsible for IL-1β release during hepatic I/R injury, we isolated primary hepatocytes and Kupffer cells, and determined the capability of IL-1β production. As expected, ATP and nigericin (potassium ionophore) induced IL-1β release in Kupffer cells, but failed to do so in hepatocytes (data not shown). Furthermore, neutrophils induced IL-1β release in a dose-dependent manner when cocultured with Kupffer cells (Fig. 8H).

AEBSF is suitable for in vivo use (17). Hence, to confirm the role of neutrophil-derived serine proteases in hepatic I/R injury in vivo, we examined the effect of AEBSF on liver injury and IL-1β release after hepatic I/R. Treatment with AEBSF significantly attenuated serum ALT levels, the liver injury (injured area and Suzuki Score Criteria ), the number of TUNEL cells, and the infiltration of neutrophils and macrophages after hepatic I/R injury (Fig. 9A–D, Supplemental Fig. 3). Furthermore, AEBSF treatment also decreased serum IL-1β levels and hepatic expression of inflammatory cytokines after hepatic I/R injury (Fig. 9E, Supplemental Fig. 4).

The major findings of this study are as follows: 1) IL-1β was released from I/R livers, and its deficiency attenuated hepatic I/R injury; 2) IL-1β deficiency reduced ROS generation, apoptosis, inflammatory cytokine expression, and infiltration of macrophages and neutrophil in I/R livers; 3) depletion of macrophages or neutrophils attenuated IL-1β release and hepatic I/R injury; 4) in vitro experiments revealed that neutrophil-derived proteases induced the processing of pro–IL-1β derived from macrophages independently of caspase-1; and 5) inhibition of serine proteases by AEBSF attenuated IL-1β release and hepatic I/R injury in vivo. These results demonstrate that the interaction between neutrophils and macrophages plays a crucial role in the pathophysiology of hepatic I/R injury, and provide new insights into the inflammasome-independent mechanism of IL-1β maturation during hepatic I/R injury.

Growing evidence indicates that IL-1β–driven inflammation plays a pivotal role in many sterile inflammatory diseases, and thus represents a therapeutic target (18). IL-1β release is regulated by two signals: the priming signal provides the synthesis of pro–IL-1β, whereas the secondary signal provides the processing of pro–IL-1β into its mature form. Recent studies revealed that the latter is mediated mainly by NLRP3 inflammasomes, which induce activation of caspase-1 (3, 19). Indeed, we and other investigators recently revealed the importance of NLRP3 inflammasomes in the pathogenesis of sterile inflammatory diseases, such as cardiovascular, metabolic, and renal diseases (4–8). Consequently, NLRP3 inflammasomes have received great attention as the prominent mechanism of IL-1β maturation. However, in some cases IL-1β release occurs independently of the inflammasomes; these cases are not well studied. To our knowledge, the present study provides the first evidence that the neutrophil-macrophage interaction promotes IL-1β maturation and contributes to the development of hepatic I/R injury independently of inflammasomes.

Previous studies have suggested that the mechanisms of IL-1β maturation depend on the type of inflammatory infiltrate at the inflammatory sites (18). Unlike inflammasome-dependent IL-1β maturation, which occurs predominantly in inflammatory sites where macrophages are mainly infiltrated, inflammasome-independent IL-1β maturation occurs predominantly in inflammatory sites where neutrophils are mainly infiltrated. However, in the current study, however, we showed that depletion of either neutrophils or macrophages reduces IL-1β release and attenuates hepatic I/R injury IL-1β in vivo, and that their interaction is required for IL-1β maturation in vitro. These results indicate that both cell types are indispensable for IL-1β maturation. The observed neutrophil-induced IL-1β release in primary Kupffer cells suggests that these cells are the cellular source of pro–IL-1β synthesis in the I/R liver. In contrast, Meng et al. (20) recently reported that Schistosoma japonicum infection activates NLRP3 inflammasomes and induces IL-1β production in hepatic stellate cells. We previously demonstrated that cardiac fibroblasts produce a substantial amount of IL-1β after cardiac I/R (4), therefore hepatic stellate cells may participate in the production of IL-1β during hepatic I/R injury.

We identified the distinct role of neutrophils and macrophages in IL-1β maturation: neutrophils release proteolytic enzymes, whereas macrophages synthesize their substrate pro–IL-1β. Previous studies identified neutrophil- and macrophage-derived serine proteinases, such as proteinase 3, neutrophil elastase, and cathepsin G, as enzymes that can process pro–IL-1β into its mature form (18). Of these, Coeshott et al. (21) suggested that proteinase 3, unlike neutrophils elastase or cathepsin G, induces effective maturation of IL-1β. In the current study, we identified Val114 and Tyr113 as the possible cleavage sites of pro–IL-1β by neutrophils. Val114 is known as the cleavage site of proteinase 3, whereas Tyr113 is known as the cleavage site of neutrophil elastase and cathepsin G (18). Thus, we propose that multiple proteases, including proteinase 3 and neutrophil elastase, may be involved in neutrophil-induced maturation of IL-1β.

Several limitations of this study should be noted. Firstly, although we demonstrated that neutrophils promote the maturation of pro–IL-1β, we did not identify the priming signals inducing pro–IL-1β synthesis in the I/R liver. Regarding endogenous priming signals, a number of signals, such as high mobility group box 1, heat shock proteins, heparin sulfate, fibronectin, fibrinogen, and hyaluronan, during I/R injury have already been reported (22). These signals are ligands for TLR 4 and induce pro–IL-1β synthesis via NF-κB activation. Secondly, several studies have investigated the role of NLRP3 inflammasomes in hepatic I/R injury (23, 24). These studies are somewhat inconsistent with our results. The contribution of inflammatory pathways may depend on the extent of liver injury and the status of inflammatory response after hepatic I/R. Thus, further investigations are necessary to elucidate the precise mechanisms underlying IL-1β release and liver injury during hepatic I/R.

In conclusion, we demonstrated that the neutrophil-macrophage interaction promotes IL-1β release and causes IL-1β–driven inflammation in the I/R liver, independently of inflammasomes. Moreover, multiple neutrophil-derived proteases that process macrophage-derived pro–IL-1β were identified, indicating that both types of cell are indispensable for IL-1β maturation. The present findings suggest that neutrophil-macrophage interaction is a therapeutic target for hepatic I/R injury, and provide new insights into the inflammasome-independent mechanism of IL-1β maturation in the liver.

We thank Naoko Sugaya, Masako Sakurai, and Minako Nakada for technical assistance, and Dr. Yoichiro Iwakura (Tokyo University of Science) and Dr. R. Coffman (DNAX Research Institute) for providing IL-1β^−/− mice and RB6-8C5 hybridoma, respectively.

The authors have no financial conflicts of interest.